G-50 mini columns

theodorn at medlib.georgetown.edu theodorn at medlib.georgetown.edu
Wed Jun 24 09:09:12 EST 1998


In article
<Pine.A41.3.96a.980622175559.89572A-100000 at dante09.u.washington.edu>,  "S.
Tan" <sltan at u.washington.edu> wrote:

>
> Hi:
>
> I recently purchased ProbeQuant G-50 Micro Columns from Pharmacia Biotech
> for purification of labeled DNA probes.  I would like to know if it is
> also possible to use these columns to separate labels from protein
> preparations.  Specifically, I want to remove free 32P-labeled ATP
> from a kinase reaction mixture so that I can use the phosphorylated
> protein product (about 70 kDa) as a substrate for phosphatase assay.  Will
> I need to re-equilibriate the column with other type buffer?  At what
> speed should I spin the column at?  Any advice/suggestion would be
> greatly appreciated.
>
> Sincerely,
> Stan
>
>

Actually, the original reference for the use of G-50 spin columns was to
separate phosphate (or was it ATP?) from F1 ATPase for the determination of
equilibrium binding constants (I can't remember the ref. off the top of my
head, but if you give me a couple of weeks I might be able to find it).  That
paper also describes the times and speeds for the spin; I seem to remember
100-200 g for 2 minutes. (For a one ml column poured in a 1 cc tuberculin
syringe, which is the way I still make them)

Also, G-50 spun columns were used to detremine the equilibrium binding
constants for peptide to Bip/GRP78 (Rothman? again, I can find the ref. if I
dig for it), which is about the size of your protein.

So, to make a short story long, yes you can use spin columns to separate
proteins for small molecules.  Of course, you can re-equlibrate the column
with whatever buffer you protein would be happy in.

Nick Theodorakis
Georgetown Univ. Med. Center

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